The present invention relates to isolated polynucleotide molecules which encode a novel neuropeptide Y (NPY) receptor designated NPY-Y7. In addition, the present invention relates to the use of these molecules in the production of NPY-Y7 receptors using recombinant DNA technology and to methods of screening and testing compounds for agoinist or antagonist activity.
Neuropeptide Y (NPY) forms a family (called the pancreatic polypeptide family) together with pancreatic polypeptide (PP) and peptide YY (PYY), which all consist of 36 amino acids and possess a common tertiary structure. NPY receptors, members of the G protein- coupled receptor superfamily, when activated influence a diverse range of important physiological parameters, including effects on psychomotor activity, central endocrine secretion, anxiety, reproduction, vasoactive effects on the cardiovascular system and strongly stimulates food consumption. Specific agonists and antagonists of NPY are therefore likely to be of substantial benefit for therapy of a wide range of clinical disorders. As NPY possess a compact tertiary structure and different parts of the molecule are required for interaction with different subtypes of the receptor, the logical developments of both agonists and antagonists is critically dependent upon the availability and knowledge of specific receptor structure.
It is presently known that NPY binds specifically to at least six receptors; Y1, Y2, Y3, Y4, Y5 (or xe2x80x9catypical Y1xe2x80x9d) and Y6. While it has been demonstrated that NPY receptors couple to the adenylate cyclase second messenger system, it remains probable that additional NPY receptor subtypes exist since there is evidence that phosphatidylinositol turnover, cations, and arachidonic acid may also function as second messengers for NPY.
Since NPY agonists and antagonists may have commercial value as, for example, potential anti-hypertensive agents, cardiovascular drugs, neuronal growth factors, anti-psychotics, anti-obesity and anti-diabetic agents, the ability to produce NPY receptors by recombinant DNA technology would be advantageous. To this end, DNA molecules encoding Y1, Y2, Y4, Y5 and Y6 have previously been isolated.
The present inventors have now isolated novel DNA molecules encoding the human and murine NPY-Y7 receptors.
Thus, in a first aspect, the present invention provides an isolated polynucleotide molecule encoding an NPY-Y7 receptor or a functionally equivalent fragment thereof.
The encoded NPY-Y7 receptor is characterised by the N-terminal amino acid sequence:
MX1X2MX3EKWDX4NSSExe2x80x83xe2x80x83(SEQ ID NO: 1),
wherein X1, X2, X3 and X4 are selected from codable amino acids but, preferably, X1 is selected from Phe and Ser, X2 is selected from Ile and Thr, X3 is selected from Asn and Ser, and X4 is selected from Thr and Ser.
More preferably, the polynucleotide molecule encodes a human NPY-Y7 receptor of about 408 amino acids or a murine NPY-Y7 receptor of about 405 amino acids.
Most preferably, the polynucleotide molecule encodes a human NPY-Y7 receptor having an amino acid sequence substantially corresponding to that shown as SEQ MD NO: 2 or a murine NPY-Y7 receptor having an amino acid sequence subtantially corresponding to that shown as SEQ E) NO: 3.
The polynucleotide molecule may comprise a nucleotide sequence substantially corresponding or, at least, showing at least 90% (more V preferably, at least 95%) homology to that shown at nucleotides 1 to 1903 or nucleotides 369 to 1592 of SEQ ID NO: 4 or any portion thereof encoding a functionally equivalent NPY-Y7 receptor fragment.
The polynucleotide molecule may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable bacterial, yeast, insect and mammalian host cells. Such host cells may be used to express the NPY-Y7 receptor.
Accordingly, in a second aspect, the present invention provides a mammalian, insect, yeast or bacterial host cell transformed with the polynucleotide molecule of the first aspect.
In a third aspect, the present invention provides a method of producing NPY-Y7 receptors or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect-under conditions enabling the expression of NPY-Y7 receptors or functionally equivalent fragments thereof.
Preferably, the host cell is mammalian or of insect origin. Where the cell is mammalian, it is presently preferred that it be a Chinese hamster ovary (CHO) cell, monkey kidney (COS) cell or human embryonic kidney 293 cell. Where the cell is of insect origin, it is presently preferred that it be an insect Sf9 cell.
In a preferred embodiment, the NPY-Y7 receptors or functionally equivalent fragments thereof are expressed onto the surface of the host cell.
The polynucleotide molecule of the present invention encodes an NPY receptor which may be of interest both clinically and commercially as it is expressed in many regions of the body and neuropeptides of the NPY family affect a wide number of systems.
By using the polynucleotide molecule of the present invention it is possible to obtain NPY-Y7 receptor protein or fragments thereof in a substantially pure form.
Accordingly, in a fourth aspect, the present invention provides a NPY-Y7 receptor or a functionally equivalent fragment of said receptor, in a substantially pure form.
In a fifth aspect, the present invention provides an antibody or fragment thereof capable of specifically binding to the NPY-Y7 receptor or functionally equivalent fragment of the fourth aspect.
In a sixth aspect, the present invention provides a non-human animal transformed with the polynucleotide molecule of the first aspect of the present invention.
In a seventh aspect, the present invention provides a method for detecting agonist or antagonist agents of an NPY-Y7 receptor, comprising contacting an NPY-Y7 receptor, functionally equivalent fragment thereof or a cell transfected with and expressing the polynucleotide molecule of the first aspect, with a test agent under conditions enabling the activation of an NPY-Y7 receptor, and detecting an increase or decrease in activity of the NPY-Y7 receptor or functionally equivalent fragment thereof.
An increase or decrease in activity of the receptor or functionally equivalent fragment thereof may be detected by measuring changes in cAMP production, Ca2+ levels or IP3 turnover after activating the receptor or fragment with specific agonist or antagonist agents.
In a further aspect, the present invention provides an oligonucleotide or polynucleotide probe comprising a nucleotide sequence of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to the polynucleotide molecule of the first aspect under high stringency conditions (Sambrook et al., Molecula Cloning: a laboratoy manuiual, Second Edition, Cold Spring Harbor Laboratory Press).
In a still further aspect, the present invention provides an antisense oligonucleotide or polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to an mRNA molecule which encodes an NPY-Y7 receptor so as to prevent translation of the mRNA molecule.
Such antisense oligonucleotide or polynucleotide molecules may include a ribozyme region to catalytically inactivate mRNA to which it is hybridised.
The polynucleotide molecule of the first aspect of the invention may be a dominant negative mutant which encodes a gene product causing an altered phenotype by, for example, reducing or eliminating the activity of endogenous NPY-Y7 receptors.
The term xe2x80x9csubstantially correspondingxe2x80x9d as used herein in relation to amino acid sequences is intended to encompass minor variations in the amino acid sequences which do not result in a decrease in biological activity of the NPY-Y7 receptor. These variations may include conservative amino acid substitutions. The substitutions envisaged are:
G, A, V, I, M; D, E; N, Q; S,T; K, R, H; F, Y, W, H; and P, Nxcex1-alkalamino acids.
The term xe2x80x9csubstantially correspondingxe2x80x9d as used herein in relation to nucleotide sequences is intended to encompass minor variations in the nucleotide sequences which due to degeneracy in the DNA code do not result in a change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not; result in a decrease in biological activity of the encoded protein.
The term xe2x80x9cfunctionally equivalent fragment/sxe2x80x9d as used herein is intended to refer to fragments of the NPY-Y7 receptor that exhibit binding specificity and activity that is substantially equivalent to the NPY-Y7 receptor from which it/they is/are derived.
The terms xe2x80x9ccomprisexe2x80x9d, xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.
Reference to percent homology made in this specification have been calculated using the BLAST program blastn as described by Altschul, S. F. et al., xe2x80x9cCapped BLAST and PSI-BLAST: a new generation of protein database search programsxe2x80x9d, Nucleic Acids Research, Vol. 25, No. 17, pp. 3389-3402 (1997).